Hi Peichuan,Nice protocol! I have a few questions about the...

Feb 17, 2012
protocol Protocol: PCR-RFLP Genotyping of Point Mutations in Caenorhabditis elegans
Hi Peichuan,

Nice protocol!

I have a few questions about the electrophoresis condition you use: Do you use regular agarose for the gel? And do you use TAE or TBE buffer? I'm deciding whether to use agarose gel or acrylamide gel, and can you give me some suggestions?
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Sophia Answered Feb 17, 2012

VT

Hi Peichuan,

Thank you so much for your detailed explanation! I'm going to try out your protocol and will let you know of my progress.

Sophia
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Peichuan Zhang Answered Feb 17, 2012

Department of Biology, The Pennsylvania State University, USA

Hi Sophia,

Thanks! I'm glad that our protocols have attracted attention from potential users.

We have been using regular agarose gel (2.0%) and 1X TAE buffer all the time. In my case, I need to separate two bands of about 200-bp (with difference of ~30-bp), and sometimes, I used 2.5% agarose gel instead. Try to not use too high voltage to avoid "smiling effects", and always remember to have positive and negative controls for comparison. Also use fresh 1X TAE buffer, as its buffering capacity drops significantly after a few rounds. You may consider designing a few different PCR-restriction digest strategies and pick the one that produces best resolution.

I only used TBE buffer, which is better than TAE in its buffering capacity, for gel shifting experiments. I recall that I saw some notes about achieving better resolution with TBE for smaller DNA (<300-bp on 2% agarose gel).

Hope this would help you a bit. Please let me know if you have further questions, and best luck with your experiments.

Peichuan
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